Benzyl alcohol-water countercurrent distribution extraction of vitamin b12



g- 1952 N. G. BRINK ET AL 2,607,717

BENZYL ALCOHOL-WATER COUNTERCURRENT DISTRIBUTION EXTRACTION 0F VITAMIN B Filed Oct. 8. 1949 2 SHEETS-SHEET 2 UNTERCURRENT DISTRIBUTION I5% MFITERIHL OF DISTRIBUTION K CwiCs) L 0F DISTRIBUTION Cw:

63 INVENTOR I NORMHN e. BRINK .2? BYFRHNK J. WOLF B W I N ATTORNEY Patented Aug. 19, 1952 .BJEN '1; ZDISERJBHTION epnietiomoanees,lsngs nnneqneasa This invehtiqn is;coneerned withanewand in pm ed: process w ic wee lla ef 11 the reco ery vita i -like ateria st om; other em c l eomneund th w ich hey he associated? "Mere-1 p rticu ar y. our i en on 5. concern d with the m ve s andesena t nr of I Buzz m m tune ub ta s which tt ievita B,124 R e .ntalQ e-wit e h hemieamcemmu ds and substan es, ome; o hic ass iat d su stances am; I at p esent th li ht a.. e-q oselnrelatedtethe.=.vitamin,.b t i chem cal} CQQSII'LZIZ-QQIL; a in nhrsie oeiea XV... 1 .1 1.11.512: is 'a new v am nzoi the B:-series .1. is .s qrvsta-ll ne iq m from liver r ported to theient fie wor dzmva eoznmmicatiQn bx R hesovBrink, Kenius n; Wood, and Eo kersenu lished in) Sn erx e n Anrik 16,, 1:348; mi: .07. naeeszfiafi and 3%.. Theiqonendina natent Dalmatian. or; Riches-and Wood,,:SeriaL No; 203 Quilted; emit 110;, 1948, abandoned, is direntedtoi this: new; vita-111.1112 and a; method fen its reeoverx;ironesouncesthereof. v.

-B1z-. oeeurs im a; number-or ma? terials; will; beanparent I from .renontt or the; autlmrsmefenred .tmale'ove in science-roamecemhen 3;.;;19AB,,V0111IIIBL1;Q85 pages 634e and; 635i However;;;due; to; its: low: eoncentration. im'these materialaxthe; separation: and: purification of vitamin Binha'si been'tfonnd to beta,- ivery difiicult problem certain fermentation: broths; rfor examplexwitamin; B'wooeurs: 1n \quantities that apparentlyoneyenr exneed one: part. in: ten million;

Such extreme dilution has naturally made meth:

ods. to isolate ithez vitamin, -:and to recover it in V substantially. pure: form; "very: difiiouit: to oarry outconcthe industria h'scaleh has; moreover, necessitatedsa wholevseri'es-ofipurification steps, and a specialized. technique -vvhieh"- is difficult to carry out whena'treating: large volumes: of source materials w -We have now found that vitamin Bm-eanbe readily; separated from: substances; that accompany itinfnaturalln ooeurring'media which substances: are apparently 'very closelyrelated, both chemically and im physiologieat action,- to' vitamin'Bm, bya process which'- involvescountereurrent distributioirof the vitamin' Bir betweerr the various constituents" of a partitions01vent;

- Among suitable partition solvents weliave found benzyl' aloohol water very satisfactory: Our'improvedc process permits theready recovery of vitamin B12 in crystalline form with 81 mini?- mumvof operationarsteps; and at more less ex+ 05m (01. rev-her pense than, is required: for; it's. recovery bynresently-available, procedures v Because of, the. very} dilute concentration of vifiaminBiz, in certain souroe frem whih, the v vitamin is obtained} such as, the. various broths prepared; by microbial; fermentati'omand because of its occurrence in those souroesalong with ao companying substanceswhich. are ,elosely, related to. vitaminBiz'; isolation andrecovery offthe vitamin in'crystalline i0rm,,a nd ina relativel y highstate of.purity hashithentoinvolved a great deal of. 'dilficulty. D e methods new employed for. recovering, vitamin v BIZ generally involve a series. of; pnrifieation steps anditheytmayrinolude adsorption. procedures. chromatographyL-and iractionai precipitation Even with these highly sp ebiali'zed techniques; when operating; on, the commercial scale with considerable. volumessogt starting material, it. has, been observed! thatmhere isrpresentpalong with the; final. product certain undesirable, materials. similar: in; constitution. to BIZ WhiChV materials crystallize along with. the. vitamin in. the final. purification steps In an efiort to,v isolate, and. recover the vitamin B12 fromthese-materials. intahigh state of purity,

it hasheen. necessary: to; utilize, a -whole series of reneated. crystallization; steps; the. produether ins-r crystallized and recry ll z domawater; or from a mixture. of water; and. an. organie-..SQ1- vent. I While such; procedures have been-successful to a limited extent in the recovery ofivitaxmin 312, andin its7 separation. fromthese other ma erials.- with wh t naa qqeurs these results, have. beensecured only withzthe; accent.- nanying, loss of a. very; considerablea-pnoportion oithe vitamin Bmpresent.

In addition to the inability of presentlyeavaih able: methods to reeoveli" all Of: the vitamin; B12 in, the various source materials treated,;these methods; have; also been. subject: to; anothengreat ohieetie in. t at hey; cannot be: relied upOnito yield the: gdesii ed; vitamim. vitamin -B'12,- in subastanti'ally pung-fomn The: methods now ava'il ablenassaz matten of faotiwillonly infreqiie ti-y yieldiazlproduct having a degreeof purity inex- 'cess' of f7? this is less t'hBJIlf'tifiB inimmum degree of purity required-forvitaminjBiz wh'eh is-intended for'use in' clihicalstudies by them dical profession as the, pure vitamin it ise] d ent that 'there iiasbeen a.wide.fiel d' for improvement in presently-available; industrial recbyry prose.- dures.

WhiIethe nature ofth Vitamifi B12-.-ljkewmat rials which; occur along, with vitamin. 1B

to the vitamin. However, in order to provide However, these activities are Thus, from the chemical and biological point of. view they appear to be closely rel'ated substantially pure vitamin B12 whichis availablefor the treatment of diseases such as permeate; anemia, it is essential that the vitamin". -B12-like materials, present along with the vitaminBiz,

be removed, and that the vitamin be supplied to 2 We have discovered that vitamin B12 can be very eificiently and efiectively separated from accompanying substances by utilizing for its recovery a countercurrent distribution procedure employing a suitable partition solvent. v By utilizing such apro'cedurel the undesired substances with which vitamin B12 is associated in naturally-occurring'sources and media remain in certain fractions of the countercurrent distribution liquid, while theivitamin B12 content of the produot undergoing; treatment is withdrawn to and concentrated in-a fraction of the partition solvent from which itmayreadily be recovered in substantially'pure crystalline form. V While ycountercurrent distribution methods have previously been used under some circumstances, it is indeed surprising that simple counthe medical profession in substantially pure,form,

i. e., as a product of 95%. purity or better.

Accordingly, it is .theprincipal object of our invention to provide a method by which vitamin B12 can be separated, in substantially pure form,

, e. glyas a product of 95% purity or higher, from other materials similar to vitamin B12 in chemical and physiological activity with which the vitamin B12 is associated in the usual source materials for the new vitamin. v

Itis another object of our invention to provide anew and improved separation step for effecting the isolation of vitamin-B12 in substantially pure form from fermentation media and other natural sources in which itis present, which method may be utilized either in conjunction with presentlyavailable recovery methods, or independently of present methods, to recover the desired vitamin iii-the substantially pure crystalline form and substantially free of these naturally-occurring vitamin B12-like contaminants. 'It 'is still another object of our invention to provide a method for the recovery of vitamin *BIZ from materials in which it maybe present, which method permits the rapid and easy separation of vitamin B12 from materials of a similar na: ture,- and of similar physiological action, with which it may be associated, which method in- 'volves relatively inexpensive countercurrent distributionlpartition) between two solvents of a two-phase partition'solvent. In carrying out this objectwe'preferably utilize, as the countercurrent distribution solvent, benzyl alcohol and water. i

' The foregoing as other-objects thereof of an auxiliary character,

tercurrent distribution can be utilized to bring about the ready separation of vitamin B12 in a high state of purity from naturally-occurring materials whichare'of generally similar chemical construction and [physiological activity. This is especially surprising when the extreme dilution in whiohthe vitamin B12 is present in the product undergoing treatment-is considered.

We have found that the best solvent mixture to employ in the countercurrent distribution for the concentration and recovery of vitamin B12 is a twoaphasesolvent mixture, preferably-a mix-' ture ofbenzyl alcohol and water, although it may be possible to substitute other solvents (such as cres'ol-toluene or cresol-canbon tetrachloride mixture for benzyl alcohol. When this solvent mixture is employed'as the partition-liquid it has been found that the undesired substances forming the largest class of impurities, present along with the vitamin B12, are distributed between thewater and-the benzyl alcohol in an apparent ratio of about 8 to l, whilethe vitamin objects of our invention, aswell will-be apparent from the ensuing disclosure of w K=8.0; and 5% of an impurity jof K=0.4. In 5 this countercurrent distribution, the resultsof which areindicated. graphically in Fig. 2,,the system benzyl alcohol-water is utilized asqthe distribution solvent. 1 H Y Throughout the specification and in the drawing, the number of transfers betweenthe .two solvent phases will be referred to either as a number of plates or as a number of. vessels.

It wil1, of course, be apparent that the term plate refers to an equilibrium stage.

out as follows:

B12 present in'the mixture undergoing treatment, on the other hand, is distributed between the Water and the benzyl alcohol in an: apparent ratio of about 1.2 to 1. By thus separating-on the benzyl. alcohol phasezof. the two-phase solvent mixture it will befound that. the vitamin B12 substantiallyfree from contaminants ispresent to the greatest extent in the benzyl alcohol phase, this phase containing. only about oneninth of the undesiredsubstances. of vitamin B12-like character which. were-initially present along .with the vitamin. The remaining eightninths of these contaminating. substances originally present has. been distributed to thewater v phase. An; additional class of impurities hasralso .been found to be present in some instances. .iThis class is of alow order of :oceurre'nceand appearsto be distributed'between:;water and benzyl alcohol in a ratioofabout 0.4 to 1. .In operatin to obtainvitamin B12 greater than pure, it is often possible to ignore this second class of impurities.

In carrying an extendeicounterourrent distribution procedure employing a-number of plates or vessels, as for" example in the eight: plate countercurrent distribution; shown in Figuredof the drawing, the actual procedural step or mode, of operation in accordance with the di ae grammaticshOWing in the drawing are carried An .initialwessel (or platey'is charged with a quantity of. water Wito which is added a quantitypfbenzyl alcoholBl As indicated in the drawing, thevitaminBm concentrate is dissolved in the water and the benzyl alcohol is then added amen-t 5. tothe solution. The procedure; however; ism-e cisel y the same when the vitamin B12i'S'=i-I-1tr0- duced in the benzyl alcohol phase ln vessel N o;

t which on a laboratory scale maybe a 'sepa-- ratoryfunnel), the mixtureof W1 and B l is thoroughly-shaken and thenallowed-to separateinto a waterlayer WI- and a benzyl al'cohoi layer BI'." At this point, the vitamin B12 andothermaterials introduced to thesystem together therewith will be distributed between'the water andbenzyl alcohol layersr 4 -Aftcr1stratifica-tion of the waten and benzyl alcohol layers m m orvessel No. I the benzylalcohol layer BI is drawnolrand added toa quantity off-reshwater W 2-- in a secondlplateor vessel No.- 2- as indicated in -the--drawing To the plate or vessel- No; I containing the- ;initial water layer Wli's-"addeda fgresh quantity ofbenzyl alcohol "B2 as indicatedyin the drawing. The two'plates or vessels now-in the' sys'temare thoroughlyshaken as above described causing a redistribution ef the vitamin B12" and associated materials in the liquid phases of the two plates or vessels. After this agitation, the bcnzyl alco-- hol; layer B'I is withdrawn from vessel No. 2 and} added toplate orvessel' No. 3 containing a fresh quantity ofwaterW3. The-'benzyl alcohol layer B2 in plate-or-vessel'No. l is Withdrawn and; added tovessel' No. 2 containing the water layer W2.

To plateor vessel No. I containing the water layer W1 isadded a fresh quantity ofbenzyl alcohol-B3; The three plates or vessels now in" the system, i. e., those identified in the drawing as containing water and benzyl alcohol layers W3-/BI'-, W2/B2-and- WI /-B3 are again shaken and allowed toseparate for redistribution of the vitamin Biz and associated substances in'the respective layers-of each plate orvessel. 'I-he ibenzyl alcohol'layer Bl fro-m .p-late-orvesselNo. 3 is then withdrawn and added to plate or' vessel No. 4 containing a fresh'po-rtion of water W4; The benzyl alcohol layer-"B2 is then transferred to vessel No; 3 containing; wateryfraction W3, and the benzylalcohol layer B3 in. Vessel; No. I transferred to vessel No. 2 containing waterfraction W2;v and a fresh quantity of" benzyl' alcohol B'qifisadded to vessel No. I containing water fraction WI After eight portions of :water and benzyl alcohol havebeen' added to the system, it will be evident; that the eight plates or vessels contain water and benzyl alcohol". layers W8/B-l, W 1/B2; W6/B3, W/B4, etc., as indicated alon the diagonal line in the; drawing.

When multiple extractions as illustrated in the flow sheet constituting Fig. 1 of the" drawing are carried out, this countercur'rent distribution procedure i s-,fin fact, so effective for the recovery of vitamin-B12 that it may be. utilized in an assay procedure for determining the amountfof the vitamin present in, various broths obt ined; by microbial fermentation, and in various intermediate solids and solutions containing vitamin Bmas secured. An assay procedure which may be utilized for the determination of vitamin B12 is given. in the copending application.oi F1, J. Serial No. 120,009,, filed October 6, 19.49,

now Patent No..2,'5.3.0,416. l

In, carrying out countercurrent' distribution utilizing-a two-phase partition solvent such as benzylalcohol and water, wehavefoundthat the processmay be carriedout on the industrialscale in a. number of ways; For: example, itisipossible utilizethe; following mod'ifiedcountercurrent distribution technique, this modified-procedurethere is then added a'fvolume of" benzyl alcohol which haswbeen saturated with -Water, wl1ich volume is equal tothat of the volume of the umter saturated with Ibenzylalcohol orig-inallyi Line troducedr- Thesolvent mixture is then agitated in order to accelerate the distribution; The l solvent mixture is'then allowed to stand, thebcnzyl alcohol layer removed 'andseparated; and this benzyl alcohol layer is th'en added to the second extraction-- vessel. Agitation of the-solvent mixture in vessel. is then effected. At theter mination of the agitation the solventini xtureis allowed to stand. Thebenzylalco-hol layerseparates, and -it is then removed and placed i=n-a suitable storagervessell r r v 3 These countercurrentextraction steps are-repeated anumberof times; usually six or more times, utilizing-eachtimefresh portions of benzyl alcohol. At the end of these operations, all-of= the benzyl alcohol layers obtained are-combined; The benzyl alcohol solution resulting from the use of a total of seven portions-of the alcohol-contains about 94% of the vitamin B12- originally. present in the material treated andonly about 20% of the undesired materials having an apparent distribution ratio of =abo-ut8'to l, chemicah ly and physiologically similar tovitamin which-were present in the source material treated: The impurities: havinganapparent distribution ratio-of about 0&4- to 1-,- when'p'resent willlbeio'und' in the benzyl alcohol solution." This solution-of vitamin B12 in benzyl alcohol of greatly improved purity may be then treated in various "wa s to bring about the precipitation of the vitamin Biz present therein. There is thus secured 'acrystalline product of'very high purity, a product-be tween 95% pure and pure often being ob tained. f

By-increasing the-number of ex-t'racti'on vessels containing water saturated with benzyl alcohol which are employed in a series, it is possible to bring about the more complete separation of the major class of undesired substances" from the vitamin B12 recovered. In order to maintain a suitable high percentageof recoveryof vitamin B12 in the product being treated, it isgenerally preferable to increase the number of ben'zyl alcohol solvent portions employed in the counter-'- ourrent distribution, and. this method is often highly advantageous in its operations, giving very satisfactory results, when the material. treated is of a relatively low degree of purity insofanzas its vitamin B12 content is; concerned; 1

Frequently, the impurities of; distribution. ratio 0.4;to; I are found in undesirably high proportions; and we therefore prefer to;- use-the'following process which will nearly'completely remove these impurities' thereby I permitting the: ohtainment of vitamin B12; which is nearly- 100%. pure. It is therefore preferred to'ordinarily carryout acom+ plete countercurrent' distribution; its-illustrated in the. flow-sheet constituting Fig. 10f the drawing. Thecontents. of the. distribution vesselsare then crystallized individually, and the. crystals of the desired purity-are segregated; The vitamin B12 of highest purity. will. be obtained from the center vessels, .while the; impuritiesofvdistribution K =8.() and K; 0..4; will be; foundv predominantly in.the flrstzseveralzvessels and thei lastsseveral 7. vessels, respectively. In this manner, substantially pure vitamin B12 is separated from the two principle types of impurities. a

In the foregoing illustrative examples we have specified that equivalent amounts of rbenzyl al cohol'and water should be utilized, butit is'also, possible, of course, to vary therelativeamounts, of benzyl alcohol and water employed in the countercurrent distribution procedure. These proportions can be varied withinconsiderab-le without interfering with the improved re sults attained. However, somevariations in the distribution of vitamin B12 and its accompany-. ing contaminants over the extraction vessels may occur,:and this may necessitate changes in the number of extraction vessels and in the ulnber of water and, benzyl alcohol portions employed for the countercurrent distribution.

Qur process is particularlyuseful for the treatment of materials which are already high in vita-- min B12 content, such as, for example, source nraterials containing. from 10% to.90% of vitamin- B12.. However, the utility of our process isby no means confined to the treatmentof materials of high vitamin B12 content, as our improved procedure may be utilized to treat materials of widely different vitamin content. It may also be utilizedfor the purpose of removing other impurities from the vitamin B12 concentrates at intermediate points in the processing of those concentrates for the purpose of recovering their vitamin I content in the form of a substantially pure, crystalline product. For example fermentation broths as secured by the fermentation of various microorganisms including Streptomyces griseus may be treated by our improvedmethod directly, and without any preliminary purificad01 However in most cases, it is ordinarily preferred to first subject fermentation broths as thtained iby microbial fermentation to various other purification procedures in order to remove the bulk of impurities present therein, and to provide a material which is fairly high in vitamin-Biz concentration, which material can then be ef-.

ficiently and effectively treated by our improved .countercurrent distribution process in order to Erample 1 v,

i-Afermentation broth resulting from the elaboration of S.-' griseus was treated with activated charcoal to adsorb the active factors. The ch'arcoalwas then eluted with aqueous pyridine to re move'these factors, and the eluate was concentrated: The active factors were transferred to benzyl alcohol, and the resulting solution was chromatographed on activated alumina; the colunm being developed with methanol. The active factors were precipitated several timesby adding acetone to a methanolic'solution; a flocculent precipitate being thereby obtained. A representative precipitate weighed 40 grams and was found to contain about 2650 milligrams of vitaminBiz-like materials by measurement of the intensity of. absorption at 5500A (a characteristicpeak'forvitaminBiz) and comparison with the known value for pure vitamin B12. I 1

Theprecipitate was dissolved in methanol and chromatographed on activatedv alumina. 'the column being developed with methanol. The red coloredrich efliuent was found t contain 2280 milligrams of vitamin B z-like materials by meas: urement of the intensity of the absorption at 5500A, The rich efliuent was concentrated and;

thenprecipitated Iwith-acetone-ether. ,Thepre this operation being carried out in a centrifuge tube. In a second centrifuge tube there; was

- placed 100 milliliters of water saturated with .benzyl alcohol 100 milliliters of benzyl alcohol saturated with water was then introduced into the first tube, andthe tube was shaken well. The tube was centrifugedv to effect separation of the water and lbenzylaloohol layers. The lbenzyl alcohol layer was removed from the bottom of the twbe and. then introduced into the second tube, where the shaking, centrifugation, and separa: tion were repeated. The benzyl alcohol layer was stored separately in a collection vessel. 'Six additional 100 milliliter portions of benzyl alcohol saturated with water were passed through both tubes in the same manner, and the-seven benzyl alcohol layers from the collection vessel were combined. v 1 3 The combined benzyl alcohol layers were concentrated in vacuo at 35 C. to remove water, and {1.2 liters of ether "and 1.4 liters of acetone were added to the cooled concentrate. A precipitate formed, and it was removed by filtration and dissolved in400 milliliters of methanol. To this solution 'there wasadded 2 liters of ether, and precipitate formed and was removed by filtration. This precipitate was dissolved in 21 millilitersof water and it was allowed to crystallize at 0-5? 0. crystallization requiring a period of about 15 hours. I The crystals that formed were removed by. centrifugatio-n, and they were. then-washed with water at 05 CQ The crystals were redissolved'in 100 milliliters of water, and the solution was then filtered through a sintered glass funnel. The apparatus was washed with 20 milliliters of water, and the wash water was combined with the filtrate. To this solution there was then added 1320 milliliters of acetone," and the resulting solution was allowed to stand ;for 1 hour at room temperature. An additiohalZelO milliliters of acetonewas then added, and the solution was cooled at 0-5 '0. for 3 hours. Crystals formed and they wereremoved by filtration and dried-in vaouo at room temperature; 'Iherewas thusobtamed 910 milligrams of crystalline material. .A sample dried to constant weight at 100? 0. lost 6.7% of its weight, this being the amount of volatiles present. The dried sample was found to be 95.1% pure vitamin B12 as verified byphase solubility analysis, and this is equivalent to. 808 milligramsof pure vitamin B12. r l I a Example 2 I 1 Crystallinevitamin B12, 4750 minimal-s we obtained as described in Example "1. The material was assayed for vitamin B12 content the countercurrent distribution assay procedurefidescribed in the copending application of J. 'Wolf Serial No; 120,009, filed'October 6, '1949,"andwas found to contain 4330 milligrams of the vitamin, equivalent to a purity of 91.3%. It was desired to further purify this material to raise the purity above P I a-eomis' The 47 50'mi1ligrams iof crystalline'material was subjected to a.-comrplete ;8-yplate; countercurrent distribution between lflom'illiliter phases of water and benzyl alcohoL-enrploying :procedures as described in1theforegoingspecificationand as illustrated firm-Fig. .1.' At the completion of the distribution, there were eight vessels, numbered 1 l -8, that contained water; and benzyl alcohol phases in -e4;1uilibriu m (eight-.portions of benzyl alcohol had contacted thelwater in vessel number I, seven had contacted that of Vessel'number 2, etc).

To each ofxtheeeightryessels was :added 125 ml. of chloroform, to transfer the 'activematerials to the water layer, and the, water layers were separated. Each benzyl alcohol layer was washed with an additional '25 milliliters of water, and eachiwash was combined with the corresponding water layer. Each water phasethusrcbtained was assayed for total solids present and for maximum vitamin "B12 content, by "measurement of ithejntensity of the absorption .at "5500 A; 'To each phase was then added 15 volumes'xdf: aceitone, and crystals formed on standin'g.- '.The crystals that formed in the solutions from vessels '3, 4, and-5 and :6. were removed filtration and dried, and each set .ofcrystals was assayed .forvitamin B12. content by the countercu'rrent distribution assay procedure. The results of the foregoing determinations are tabulated below:

The crystals from vessels 3, 4, and 5 were of the desired high degree of purity. The materials from vessels l and 2, containing almost all of the undesired substances having an apparent distribution ratio of about 8:1 (CwzCe), and the materials from vessels 6, l and 8, containing almost all of the undesired substances having an apparent distribution ratio of about 0.4:1 (CwzCe), were combined with other like fractions for further purification by again subjecting them to countercurrent distribution.

' The separation effected by the foregoing procedure is graphically illustrated in Fig. 2, wherein there are plotted the concentrations of the components of a typical vitamin B12 concentrate for each plate after an eight-plate countercurrent distribution is carried out. The initial concentrate is, for purposes of illustration, assumed to contain 80% of pure vitamin B12 (K=1.2), 15% of material of K=8.0, and 5% of material of K =0.4. It is apparent that the highest concen trations of vitamin B12 are found in the center plates, which at the same time contain the lowest concentrations of the undesired impurities. Plate 4, for example, contains 24.17% of the weight of the starting concentrate; of this, 23.3% is vitamin B12, 0.45% is material of K=8.0, and and 0.42% is material of K =0.4. Thus, 96.5% of the substances present in solution in plate 4 is vitamin B12. On crystallization from this plate in the manner described above, vitamin B12 at least 96.5% pure is obtained, this crystallization often efiecting some additional purification. On the other hand, it is apparent that the least vitamin E WhilezVarious 1 10 iB12,;and the mostof the. undesired materials are :found in thefirst several and last several plates.

1 It -will.,-be understood that the proportions of interfering materials present will vary somewhat with the'concentrate treated, and furthermore, that theapparent distribution-coeflicients forthe principal classesmf impurities are not absolute, lbut approximate-values Thus, the amounts of impurities found in v the center plates wil1- .of;ten 'be 1less'than-shown in Fig. 2 and may, at times, be greater. I .In the latter event; repetition 10f the distribution pmcess will provide vitamin B12 {or the desired purity. Alternatively, :a' more complete-separationcan-be effected-in one operation by increasing the number of platesemployed; .A

graphic representation-of this alternate procedure wouldshow-the-curvesrfor K='8..0 and-Kama, as 'sh'ownin Fig. .2, moving 5,110 the left and might, respectively, laway from ;the peak :of :the 1011MB -f0r:-K "1.2. '1 11 changesandmodifications may be made in our process 'asdescribed herein [with referenceto certain: preferred -;:embodiments thereof, it is intended that such .changes :and

,modifications'to the extent that they are with- :in thescope of the.iappefidedeclaimsoshalhbie con- Sideredaspartofiour invention. 5

"@Wecl'ai'mf WY 1. The process for purifying vitamin B12 that comprises equilibr'ating a starting mixture of vitamin B12 and various contaminants in a two-phase solvent consisting of benzyl alcohol and water by agitatingthe resulting mixture and stratifying the solvent phases, thereby effecting a distribution of the vitamin B12 and contaminants between the two phases, individually and successively contacting and equilibrating each of said phases with additional portions of the other phase in a countercurrent manner, thereby obtaining a number of portions of benzyl alcohol andportions of Water each containing vitamin B12 and contaminants in a vitamin B12-contaminants ratio which is different from that prevailing in each other portion and in said starting mixture.

2. In a process for purifying vitamin B12 the steps that comprise equilibrating a starting mixture of vitamin B12 and various contaminants in a two-phase solvent consisting of benzyl alcohol and water in a suitable vessel by agitating the resulting mixture and then stratifying the solvent phases, thereby effecting a distribution of vitamin B12 and the contaminants between the two phases,

drawing off the lower benzyl alcohol layer from said vessel and adding the same toa second vessel containing a fresh portion of water while adding a fresh portion of [benzyl alcohol to the water layer in said first vessel and equilibrating the mixtures thus obtained, then drawing the benzyl alcohol layer from the second vessel, advancing the benzyl alcohol layer from the first vessel to the second vessel and adding a fresh quantity of benzyl alcohol t0 the first vessel and again equilibrating the mixtures in said vessels, thereby obtaining a number of portions of benzyl alcohol and portions of water each containing vitamin B12 and the contaminants in a vitamin B12-contaminants ratio which is different from that prevailing in each other portion and insaid starting mixture.

3. The process as defined in claim 2 wherein approximatelyequal volumes of water and benzyl alcohol are employed in each equilibration and wherein additional vessels containing fresh portions of water and additional portions of fresh benzyl alcohol are added to the system until there 11 are in thesystem about 8 vessels having'benzyl alcohol and water layers in'equilibrium rangin from exhaustively extracted benzyl alcohol in contact with freshwater to exhaustively extracted water in contact with fresh benzyl alcohol, thereby effecting a'concentr'ation of vitamin B12 in the intermediate vessels;

'4. The process :as defined in claim 2 wherein approximately equal volumes of 'Water and benzyl alcohol are employed in each equilibration and wherein additional vessels containing fresh portions-of water and additional portions of fresh benzyl alcohol are added to the system until there are inthe system about 8 vessels having benzyl alcohol and water layers in equilibriumranging from exhaustively extracted benzyl alcohol in contact with fresh water to exhaustively extracted water in contact with fresh benzyl alcohol, thereby effecting a concentration of vitamin B12 in the intermediate vessels, and purified vitamin B12 is recovered from the two-phase solvent mixture in said intermediate vessels by addition of chloroform' to transfer all of the vitamin B12 to the aqueous phase and crystallization of vitamin B12 from the aqueous phase by addition of acetone.

5. The process as defined in claim 2 wherein two portions of water are contacted successively with 6 to 8 portions of benzyl alcohol and said portions of benzyl alcohol arecom'bined for retwo portions of water are contacted successively with 6 to 8 por-tions'of benzyl alcohoLsaid benzyl alcohol portions are combined and concentrated, and purified vitamin B12 is precipitatedtherefrom by the addition of ether,

NORMAN innvxfi Wong:

REFERENCES CITED I The following references are of reoordfin the file of this patent: v p

UNITED STATES PATENTS Number Name Date i 2,379,644 'Shelton ul s, 945

OTHER'REFERENCES Rickes, Science-Apr. 16, 1948, vol. 107,: pages 396, 397; 1 Rickes, Science, Dec; 3, 1948, vol. 108, pages 634, 635.

Craig, Journal of Biological Chemistry, vol; l61 V (1945) pages 321 to 332. V

Ellis, J. Pharm. and PharmacoL, vol'."1,1 p g.es

I so, 61 (1949). 

1. THE PROCESS FOR PURIFYING VITAMIN B12 THAT COMPRISES EQUILIBRATING A STARTING MIXTURE OF VITAMIN B12 AND VARIOUS CONTAMINANTS IN A TWO-PHASE SOLVENT CONSISTING OF BENZYL ALCOHOL AND WATER BY AGITATING THE RESULTING MIXTURE AND STRATIFYING THE SOLVENT PHASES, THEREBY EFFECTING A DISTRIBUTION OF THE VITAMIN B12 AND CONTAMINANTS BETWEEN THE TWO PHASES, INDIVIDUALLY AND SUCCESSIVELY CONTACTING AND EQUILIBRATING EACH OF SAID PHASES WITH ADDITIONAL PORTIONS OF THE OTHER PHASES IN A COUNTERCURRENT MANNER, THEREBY OBTAININING A NUMBER OF PORTIONS OF BENZYL ALCOHOL AND PORTIONS OF WATER EACH CONTAINING VITAMIN B12 AND CONTAMINANTS IN A VITAMIN B12-CONTAMINANTS RATIO WHICH IS DIFFERENT FROM THAT PREVAILING IN EACH OTHER PORTION AND IN SAID STARTING MIXTURE. 